Nucleic Acid Metabolism in Regenerating Rat Liver VII. Effect of X-Radiation on Enzymes of DNA Synthesis"

F. J. BoLLUM,tJ. W. ANDEREGG,ÃŽANNEB. McELYA, ANDVANR. POTTER

(McArdle Memorial Laboratory, University of Wisconsin, Madison 6, Wis.)

SUMMARY The effect of x-radiation on the appearance of thymidine kinase and DNA poly merase in regenerating rat liver was studied as a function of dose (375,750, and 1500 r) and time of administration of radiation (6 or 16 hours) relative to the time of partial hepatectomy. When the radiation was given 6 hours after operation, all levels of radiation pro duced essentially complete inhibition of the increase in enzyme levels that would be expected to occur between 6 and 24 hours after partial hepatectomy. When the radiation was given 16 hours after operation, a time previously shown to completely block DNA synthesis in regenerating liver in vivo, the usual increase be tween 16 and 24 hours was not prevented. The results are discussed in relation to data obtained on whole animals, and it is suggested that inhibition observed in in vivostudies with radiation given 16 hours after operation must be explained in terms other than the inhibition of thymidine kinase or DNA polymeraseor their formation, while radiation given 6 hours after operation may act by preventing the formation of the appropriate enzyme-forming systems.

There is now general agreement that interfer Lancker, and Potter studied the effect of x-radia ence with DNA1 synthesis is one of the important tion on DNA synthesis in vivo (1) and showed the biological effects of x-radiation. Recent work by effect of doses from 375 to 3,000 r given at various Ord and Stocken (17) and by Lajtha et d. (11, 12) times during the course of regeneration. Bollum has emphasized that the rate of biosynthesis of and Potter studied two of the individual enzymes DNA may be limited by radiation in a variety of concerned with DNA synthesis and showed that ways. thymidine kinase and DNA polymerase appear in We have been concerned with the biochemical increased amounts in the high-speed supernatant mechanisms that regulate DNA synthesis, using fraction of the liver cytoplasm at the time that regenerating rat liver as the test system. Beltz, Van DNA synthesis begins in vivo (4). The present re port shows the effect of x-rays on these enzymes in * Supported in part by a grant (C-646) from the National relation to dose and the time of administration of Cancer Institute, National Institutes of Health, U.S. Public Health Service. radiation. t Postdoctoral Fellow, U.S. Public Health Service, 1956- In the experiments reported here, DNA poly 1958. merase was studied with the use of unirradiated Present address: Biology Division, Oak Ridge National primer DNA, and thymidine kinase was studied Laboratory, Oak Ridge, Tenn. on the basis of thymidine phosphate accumulation ÃŽPresentaddress: Depts. of Zoology and Physics, Univer in the absence of primer. By administration of sity of Wisconsin, Madison, Wis. radiation at two selected times after partial hepa 1The following abbreviations are used: DNA, deoxyribonucleic acid; RNA ribonucleic acid; r, roentgens; TMP, thymi- tectomy and analysis at a time when the enzymes dine-5'-phosphate; TOP, thymidine diphosphate; TTP, thy normally appear, it was possible to demonstrate midine triphosphate; dGMP, deoxyguanosine-5'-phosphate; an interference with enzyme formation by early dAMP, deoxyadenosine-5'-phosphate; dCMP, deoxycytidine- irradiation (6 hr.) at the lowest dose (375 r) and S'-phosphate. a lack of such interference at later times (16 hr.) Received for publication August 10, 1959. by irradiation with the highest dose (1500 r). 138

MATERIALS AND METHODS alkali, and filtered onto a 1 X 3 cm. Dowex-1- The technics used for partial hepatectomy, the formate column. The column was washed with care of the animals, their irradiation, and the water to remove unchanged thymidine-H3 and preparation of the rat liver supernatant fraction neutral degradation products, and further step- have been described (1, 4). In the last experiment wise elutions with formic acid concentrations up of the present series the irradiation procedure was to 1.0 M (4) removed acidic degradation products changed to accommodate the larger number of formed in minor amounts. Thymidylic acid was animals. Twelve rats were irradiated simultane then eluted in about 40 ml. of 2 M formic acid. ously in a round, plastic cage divided into twelve Suitable aliquots of the eluted fractions were plat sectors. The cage revolved at 1 r.p.m. under the ed and counted in windowless flow counters. Total x-ray beam. radioactivity in the 2 M formic acid fractions was The enzyme assays were of three varieties, in an used as a measure of thymidine kinase. attempt to localize the biochemical lesion. Pre DNA polymerase assay.â€”Thismethod requires liminary studies showed inhibition of the over-all the presence of primer DNA and radioactive TTP, reaction in which thymidine is incorporated into together with the triphosphates of the other three DNA, previously shown to occur in the soluble deoxyribosides. It has been described elsewhere as high-speed supernatant fraction of rat liver (3). the "kinase-independent" assay or Method III (3). This over-all reaction includes the operation of at Kinase-dependent assay.â€”Thismethod requires least three separate enzymes: thymidine kinase, the use of primer DNA but utilizes radioactive thymidylic kinase, and DNA polymerase. The thymidine as the substrate. It is able to utilize the first two reactions require the presence of ATP but monophosphates of the remaining three deoxy not DNA, and the last reaction requires primer ribosides, since these are phosphorylated in the DNA plus the deoxyriboside triphosphates of all presence of ATP. The reaction depends on thymi four bases found in DNA. In the present study, dine kinase, TMP kinase, DNA polymerase, and no attempt was made to study thymidylic kinase the kinases for dGMP, dAMP, and dCMP, but individually, but it will be evident that the over the limiting reaction appears to be thymidine all reaction thymidine â€”¿Â»DNAmust include the kinase. This system has been described as Method operation of this enzyme, since TMP must be con I (3). It serves as an independent check on the re verted to TTP before it can be incorporated into sults obtained with the more specific thymidine DNA Ã•2). kinase assay described above. Thymidine kinase assay.â€”When thymidine is incubated with ATP in the absence of primer RESULTS DNA, a mixture of TMP, TDP, and TTP ac In the first experiment three tissues were cumulates under the conditions described earlier studied, and the kinase-dependent assay was uti (3). The sum of these products will be a measure lized. Radiation was administered at 14-15 hours of thymidine kinase, because the rate of their for after operation, and the animals were sacrificed at mation will be limited by this enzyme; if, in addi 25 hours after hepatectomy. The unexpected find tion, TMP kinase is present there will be more ing was that there was no inhibition of thymidine- TTP and less TMP, while if it is limiting there H3 incorporation into DNA in extracts of regener will be more TMP and less TTP, but the total will ating liver, whereas enzymatic activity of thymus be a measure of thymidine kinase unless this enzyme and spleen was markedly inhibited, indicating that is strongly inhibited by one of the products. For the radiation had been effective. From in vivo the present study, column separations of the reac studies (1) irradiation at 15 hours would be ex tion products from thymidine were simplified by pected to give almost complete inhibition. The hydrolysis with l N HC1 to convert any TDP or second and third experiments summarized in TTP back to TMP, which could then be eluted Table 1 suggested that the effect was more de with 2 Mformic acid, thereby avoiding the use of pendent on time after hepatectomy than it was on ammonium formate and problems of increased dose. It should be emphasized that all the experi self-absorption on the plates used for measuring ments summarized in Table 1 were performed radioactivity. The reaction mixture was as previ under conditions which give marked inhibition of ously described, including the final volume of 125 DNA synthesis as measured in vivo (1). It is ap H\. (4). After 10 minutes' incubation at 37Â°C.the parent that complete inhibition of enzyme activity reaction was stopped by adding 6 N HC1 to a final was obtained only when irradiation was given by concentration of 1 N, and the solution was placed 11-13 hours after hepatectomy, i.e., several hours in a boiling water bath for 10 minutes. The solu before the end of the sensitive phase (up to 18 tion was then diluted to 30 ml., neutralized with hours) deduced from in vivo experiments.

Experiment 4 was a larger experiment designed affect the activity of either enzyme if it has already to examine the dose and time dependencies of the been formed but that the formation of these en inhibition of the thymidine kinase and the DNA zymes is prevented or delayed. This suggestion polymerase. The over-all assay was also carried may be strengthened by a comparison of the pres out as a check of internal consistency. Three dif ent results with earlier work, and an analysis of the ferent doses of radiation were administered to one dose-response curves obtained. It may be estimated group of animals at 6 hours, and to a second group from the earlier data ([4], Table 2, and unpub at 16 hours. From the previous in vivo studies (1) lished), that the values for thymidine kinase and it could be expected that both groups would suffer polymerase prior to 18 hours following operation essentially complete inhibition at the 1500-r dose are roughly 1-10 per cent and 20-30 per cent of level. The results of the enzyme assays (Table 2 their respective 24-hour values. Irradiation at 6 and Chart 1) confirmed the preliminary experi hours with 750 or 1500 r results in enzyme levels ments of Table 1 in that inhibition was observed at 24 hours comparable to unirradiated 6-hour when radiation was given at 6 hours and enzyme levels. It would appear that preformed thymidine activity measured at 24 hours, but no inhibition kinase and polymerase are not inactivated by ir resulted from irradiation at 16 hours. All three radiation but that further increases are prevented assays were affected by radiation at 6 hours, al or delayed. though the dose dependence of the kinase assay It seems highly probable that the critical period was more marked than that of the polymerase (as measured by x-ray sensitivity) for thymidine assay. The over-all assay agreed with the kinase kinase and DNA polymerase ends before 15 hours, assay, indicating that the kinase was the rate- whereas their appearance in the soluble fraction limiting step. occurs in the period of 18-40 hours. Further re search on the technical problems of measuring DISCUSSION total amount of enzyme on a whole-cell basis and The significance of the present in vitro work lies in cell fractions will be needed before this point in two comparisons that may be made. The first is can be studied constructively. a comparison between present enzymatic assays A discussion of the biochemical lesions brought and earlier in vivoresults (1), while the second con about by x-radiation in terms of variations in sen cerns the time of administration of the radiation sitivity at different periods in the mitotic cycle (8) (relative to the time of operation) as it affects the and in terms of actual biochemical processes is enzymatic results. The in vivoresults demonstrate facilitated by the concept of a presynthetic period effects on the over-all process of DNA synthesis, (GÃ¬),during which DNA is not being synthesized while the in vitro results are directed toward the but in which irradiation can prevent or delay the quantitative analysis of individual enzymes. onset of synthesis, and a synthetic period (S) The first comparison shows that the inhibition which is dependent upon at least two components of DNA synthesis observed earlier in vivo, which (11), of which one (Si) is sensitive to low doses of reached 96 per cent at about 1500 r given between radiation (i.e., under 1000 r) and one (82) is re 15 and 17 hours post-operatively and analyzed at vealed in the immediate mitotic cycle only by Ã•50hours(1, Charts 1, 3) is in marked contrast to higher doses of radiation (i.e., 1000-10,000 r). It the failure of the same dose to produce demon has been suggested that the 82 component is "the strable results upon thymidine kinase, thymidylic integrity of the DNA template" with a dose-re kinase, or DNA polymerase when given at the sponse curve "suggesting a single hit type of same postoperative time (Table 2). Although it mechanism" (11). It seems likely that the GÃ¬and would be possible and desirable to carry out the Si components may be multiple in nature and may in vivo and in vitro experiments on the same ani vary in importance in different tissues. Thus, the mals as has been demonstrated in other studies immediate effects of radiation on DNA synthesis (4, 7), the effect of the 1500-r dose is consistent in thymus (15-17, 19, 20) may be due to an inhi and reproducible in both systems (probably be bition of nuclear ATP generation as an Si compo cause it is well above the dose required for a nearly nent (17), but such an effect cannot be claimed for maximal effect, as shown in Table 2). liver, which is reported to be unable to carry out The second comparison (Table 2) shows that the reaction (17). doses of radiation that fail to influence the activity In the regenerating liver system the available of thymidine kinase or DNA polymerase when the data suggest that the dose-response curve for in radiation is at 16 hours after operation are highly vivo effects is multiple in nature. The present data effective if administered at 6 hours after operation. suggest that low doses given early in the GÃ¬period This result implies that the radiation does not are able to exert an effect by preventing the syn-

Exp.123No. rata3(plus (r)7503000 (hr.)14-1511-13 studiedthymidine-H3->DNAthymidine-H3->DNA

3 controls)3 Thymus Strong SpleenLiver StrongStrong

3 750750Time* 14-1512Reactionthymidine-H3â€”DNAthyimdine-H3->DNALiverLiver NoneStrong (plus 3 controls)6

(plus 6 controls)Dose TTP-H3->DNATissueLiver LiverInhibitionNoneStrong

* Time in hours after partial hepatectomy. Animals were always sacrificed at 24-25 hr.

TABLE 2 ENZYMEASSAYSONS8FRACTIONFROMIRRADIATEDRATSWITHREGENERATING LIVERS,BASEDONTHYMIDINE-H*(EXPERIMENT4) Three rats were used at each x-ray dose, and each rat was assayed by all three methods. Results for individual rats are given (in the same order for each assay), together with the average for each group.

ASSAYIrradiation

6 hours after operation: Kinase assay phosphates)Polymerase(counts/min in thymidine 51,000 3,900 900 600 68,00060,0007,60015,0006,4002,1004,4002,1002,6007006301,500

assay DNA)Over-all(counts/min/mg 11,700 2,100 1,900 1,900 8,0009,10010,7004,2002,8001001,7002,100240 1,7001,700120

assay DNA)Irradiation(counts/min/mg 9,200 700 210 50 9,1009,70035,0002,4001,10068,00076040045,000 20012042,000

16 hours after operation: KinaseassayPolymerase 9,500 7,100 32,000 32,000 44,00030,0005,40035,00037,0008,30036,00038,0009,20044,00039,0009,700

assay 3,000 3,800 9,200 10,200 7,6005,3006,6004,3005,5008,9008,9009,1004,4008,7009,5007,000

Over-all assay062,000 2,300 1,350 8,000 3,100 7,3005,400DOSE3754005,1005,100007501,0006,0006,1001.5006005,1005,100

thesis of key enzymes required for DNA synthesis. The evidence suggests that the synthesis of The results of irradiation at later times must be several enzymes necessary for DNA synthesis is explained on some other basis, since the appear prevented by x-radiation given in the early part ance of the required enzymes is unaffected by of the GÃ¬period.Evidence that may bear on the radiation. It is tempting to speculate that in both formation or release of enzyme-forming systems cases the radiation effect is the result of interfer during this period may be forthcoming from ence with DNA function, the early effect being studies on the inhibition of nuclear RNA synthesis due to the interruption of genetic function (forma by ultraviolet or x-radiation in vitro (5, 6, 13), tion of enzyme-forming system) and the later re while data showing that amino acid analogs given sult due to a destruction of the priming ability of at 10 hours following operation interfere with the DNA molecule. A recent report by Van DNA synthesis in regenerating rat liver at 24 Lancker (21) may support this postulate. hours has been obtained by Schneider et al? The prevention of the synthesis of thymidine The various biochemical lesions now available kinase by x-radiation would support the in vivo as possible sites of radiation damage are in need of result of Jaffe et al. (9), who found increased further, more systematic studies which combine amounts of deoxyribosides in regenerating liver of the in vivoand in vitro approaches. Further studies irradiated rats, and may be related to the report on the effect of subiethai doses of radiation at vari by Parizek et al. (18), who found deoxycytidine ous times in the GÃ¬andSi periods, and in terms of excreted in the urine of irradiated rats. The fact the mitotic apparatus (10) as well as in terms of that the latter compound may be deaminated only the individual enzymatic steps leading to DNA as the monophosphate seems highly significant synthesis, may be combined advantageously with (14). 2J. H. Schneider, personal communication.

1.00 0.80 0.60

0.40

g 0.20 O. K O ^ 0.10 - 0.08 uj 0.06

0.04

UJ o: KINASE 0.02

0.01 200 400 600 800 1000 1200 1400 1600 X-RAY DOSE (ROENTGEN) CHART1.â€”Effectof x-ray dose given at 6 hours after par in the period 6-16 hours after the operation. Data on the x-ray tial hepatectomy on the kinase and polymerase enzymes and inhibition of the in rico incorporation of orotic acid-C14 into on the over-all incorporation of thymidine-H3 into DNA, as DNA of regenerating rat liver are shown for comparison. The measured by in vitro assays at 24 hours after the operation. latter data are from Beltz, Van Lancker, and Potter (1), The data are not corrected for the levels occurring in the tissues Table 2, column 3. O.A. = orotic acid; TDK = thymidine.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1960 American Association for Cancer Research. Nucleic Acid Metabolism in Regenerating Rat Liver: VII. Effect of X-Radiation on Enzymes of DNA Synthesis

F. J. Bollum, J. W. Anderegg, Anne B. McElya, et al.

Cancer Res 1960;20:138-143.

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